Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting ec onomically important crops (e,g,, grapevine, citrus, and coffee). The citru s variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pat hogenic bacterium for which the genome sequence was determined. Plasmids fo r the CVC strain of X. fastidiosa were constructed by combining the chromos omal replication origin (oriC) of X. fastidiosa with a gene which confers r esistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment co mprised the dnaA gene as well as the two flanking intergenic regions, where as in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN int ergenic region, which contains dnaA-box like sequences and AT-rich clusters . In plasmid p16K, no oriC sequence was present. In the three constructs, t he promoter region of one of the two X, fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of k anamycin resistance in X. fastidiosa, Five CVC X. fastidiosa strains, inclu ding strain 9a5c, the genome sequence of which was determined, and two stra ins isolated from coffee, were electroporated with plasmid p16KdAori or p16 Kori, Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but no t when electroporated with p16K, Southern blot analyses of total DNA extrac ted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination, To our knowledge, this is the first re port of stable transformation in X. fastidiosa, Integration of oriC plasmid s into the X. fastidiosa chromosome by homologous recombination holds consi derable promise for functional genomics by specific gene inactivation.